Abstract This proposal examines mechanisms that regulate how mitochondria move within cells. Mitochondria are actively transported in all cells including neurons. Disrupted mitochondrial trafficking has been linked to axonal degeneration, dysfunctional synapses, and the pathologies of neurodegenerative disorders, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease. These studies illustrate the critical nature of maintaining the proper regulation of mitochondrial trafficking for neuronal health. Miro, also called RhoT1/2, is an essential protein for mitochondrial motility. Miro is a mitochondrial outer membrane protein with two GTPase domains that are often presumed to be regulatory switches but whose functions have remained obscure despite over a decade of study. Analysis of this protein has been confounded by several factors: 1) interpretation of overexpression studies is complicated by the presence of endogenous Miro; 2) there are two potentially redundant isoforms of Miro in mammalian cells; and 3) removal of both isoforms of Miro is lethal. To gain a better understanding of the regulation of mitochondrial trafficking, I have misdirected Miro to peroxisomes and found that it can confer on the normally stationary peroxisomes the ability to be transported by the motor complex normally found on mitochondria. By mutating the first GTPase domain of Miro, I have discovered that it regulates the ability of the Kinesin-1 motor to associate with the Miro complex. I now propose the following aims: 1) To investigate further the effects of Miro and its GTPase domains, including examining the mechanism by which it regulates kinesin binding; 2) To test competing hypotheses for how cytosolic Ca2+ regulates mitochondrial motility, and 3) To determine whether the Miro-containing motor/adaptor complex is responsible for the enrichment of mitochondria at synapses. This proposal thereby seeks to provide insights into the fundamental processes by which a neuron distributes mitochondria to support the energetic needs of all its parts. !